World Journal
of Microbiology
& Biotechnology
10, 462-464
Optimization of culture conditions for the production of rifamycin oxidase by Curvularia luna ta U.C. Banerjee Maximum activity (8.9 IU/ml) of rifamycin oxidase in Curuuhia Zunata, grown in shake-flask culture at 28°C and pH 6.5, was after 96 h. Nearly all the glucose was used in 72 h. An initial culture pH of 6.5 and 28°C were optimum for the growth and enzyme production. Among various carbon and organic nitrogen sources, carboxymethylcellulose and peptone were the most effective for enzyme yield. The rate of enzyme production was enhanced when yeast extract was also added to the medium. The optimum medium for the production of rifamycin oxidase contained 10 g each of yeast extract, peptone and carboxymethylcellulose/l and 0.04 % (NH,l,SO,. Key words: Carboxymethylcellulose,
Curvuluriu
lunata, optimization,
Rifamycin S, the starting material for the synthesis of many clinically-used rifamycins (Sensi & Thiemann 1967), is obtained by chemical oxidation of rifamycin B followed by acid hydrolysis (Seong & Han 1982). This chemical method has many disadvantages, such as the requirements for low pH and acid-resistant equipment, vigorous foaming during the reaction and, above all, the process yield is low. Rifamycin oxidase transforms rifamycin B to rifamycin S and three rifamycin oxidase-producing organisms have been reported: Monocillium (Han et al. 1983), H~micola (Seong et al. 1983) and Curvulariu Zunutu (Vohra et al. 1989). The enzyme produced by both Monocillium and Humicolu spp. is intracellular but that of C. lunutu is extracellular and has a high specific activity (Banejee & Srivastava 1993). The present study is of the effect of environmental factors (pH and temperature), carbon and nitrogen sources and growth factors on the growth of and rifamycin oxidase production by C. lunutu.
Materials
and Methods
Chemicals Low viscosity carboxymethylcellulose (CMC) was from Sigma. Different growth factors and organic nitrogen sources were
The author is with the Biochemical Engineering Research and Process Development Centre, Institute of Microbial Technology, Post Box 1304, Sector 39-A, Chandigarh 160 014, India; fax: 91 172 40985. 0
7994 Rapid Communications
462
World Journal
of Oxford
ofMicrobiology
Ltd
6 Biotechnology, Vol 10, 1994
rifamycin
oxidase.
obtained from Hi-Media Laboratories, Bombay, India. All other reagents used were of analytical grade. Microorganism and Culture Conditions Curvulariu lunutu (MTCC X5), maintained on potato/dextrose/ agar (I’DA), was cultured in 500-ml flasks, each with 100 ml of YPD medium containing 10 g each of yeast extract, peptone and dextrose/l and incubated on a rotary shaker (200 rev/mm), usually at 28°C for 7 days. For inoculation, spores were aseptically taken from a 7-day-old plate and suspended in 0.85% sterile NaCl solution and 5 ml added to the medium. Samples were taken every 24 h and analysed for cell mass, enzyme activity, extracellular protein and residual glucose. Flasks were also incubated at 26,30,32 and 34°C. Experiments on the effect of initial cultivation pH were from pH 4 to 8 at intervals of 0.50 pH units. Assay Methods Rifamycin oxidase activity was determined by measuring the concentration of the product (rifamycin S) spectrophotometritally (Seong et al. 1983); 0.025 ml enzyme solution was added to 0.5 ml of pre-equilibrated rifamycin B solution (2 IIIM, in 0.1 M phosphate buffer, pH 6.5). After incubation at 50°C for 1 h, 4.5 ml methanol/O.1 M phosphate buffer, pH 7.8 (1 : 1 v/v) was added and the reaction mixture boiled for 3 min, centrifuged for 5 mm at 2000 xg and the absorbance of the supematant measured at 525 nm. One unit of enzyme activity was defined as the equivalent of one umol rifamycin S formed in 1 h at 50°C. Glucose was determined by the dinitrosalicylic acid method and extracellular protein by the Folin-Lowry method. Dry cell weight of the mycelium was determined after filtering through Whatman (No.1) filter paper, washing thoroughly (three times) with distilled water and drying overnight at 95°C.
Rifumycin oxidusein Curvularia
lunata
Results and Discussion Effects of Culture Conditions Maximum growth of C. lunafa was at 3O”C, although maximum enzyme yield was significantly higher at 28°C (Table 1). Using 28°C for further incubations, the optimum initial pH for enzyme production was 6.5 (Table 2). The organism grew well in YPD medium at pH 6.5, with maximum cell mass (7.8 g/l) coinciding with maximum enzyme activity (8.9 II-J/ml) after 96 h (Figure 1). Glucose was all used within 72 h.
Effect of Carbon and Nitrogen Sources CMC was the most effective carbon source (Table 3) for rifamycin oxidase synthesis, giving a maximum enzyme activity of about 15 ILJ/ml. The pellet type of growth was more prevalent than the mycelial type with substrates other than CMC. One reason for the relatively high enzyme activity with CMC-enriched medium may be the relatively high viscosity of this medium; this can favour mycelial growth (Banejee 1993). Secretion of enzyme from the mycelial type of growth was better than from the pellet type. Of various organic nitrogen sources, peptone and peanut meal were the most effective
Table 1. Effect and substrate
ot temperature on growth, utilization by C. /unata.*
Incubation temperature w
Maximum cell mass (g dry WI)
Maximum enzyme yield (lU/ml)
26 20
0.3 8.0
4.1 0.2
30 32
9.1 8.2 8.4
34
enzyme
yield,
Maximum enzyme productivity (1Ull.h)
w 91 94
4.7
65
92
2.1 1.5
29 13
92 92
Initial PH
4.0 5.0 6.0 6.5 7.0 8.0 8.5
enzyme
Maximum cell mass (g dry WW
Maximum enzyme productivity (1Un.h)
yield (W/ml)
yield, productivity
6.0 7.0
0 4.0
41
6.6
6.1 8.9
05 93
7.0 8.0
a.5 8.6
8.2 1.0 0
*The organism was grown in medium containing peptone and dextrose/l, at 26°C for 6 days.
0
85 25 0
1. Course
of fermentation
ot C. lunata
peptone and &-glucose;
in medium
dextrose/l, A-enzyme
containing
at pH 6.5 and activity;
Substrate conversion
56 05
ot cultivation pH on growth, utilixatlon by C. /unata.*
Figure
10 g each of yeast extract, 28°C. M-Cell mass; II--extracellular protein.
time (h)
productivity
* The organism was grown in medium containing 10 9 each of yeast extract, peptone and dextrose/l, at an initial pH 7.0, for 6 days.
Table 2. Effect and substrate
Fermentation
conversion W
Table 3. Effect of carbon and organic oxidase production by C. lunata. Maximum activity Carbon
source
10 9 each of yeast extract,
enzyme (IU/ml)
source
on rifamycin
Maximum productivity
enzyme (1Ull.h)
(10 g/l)
Carboxymethylcellulose
15
102
&MC) Cellulose Glycerol
6.5 6.0
39
Glucose Starch
0.7 5.2
91 72
Maltose Fructose
5.6 5.5
77 76
Sucrose Molasses
7.0 8.6
97 90
Nitrogen
source
31
(10 en)+
Peptone
15
Casein
95 95 97 95 94 92 93
nitrogen
102
5.0
Tryptone Peanut meal
7.0 16.6
Soybean meal Polypeptone
12.2 11.0
Soy peptone Control (CMG and yeast
30 46 115
7.0
extract)
2.2
05 66 54 23
* Tested in medium containing 10 9 each of yeast extract and peptonell, at pH 6.5 and 28°C and for up to 6 days. 7 Tested in medium containing 10 9 each of yeast extract and CMCII, at pH 6.5 and 26°C and for up to 6 days.
World @maI
of Microbiology
6 Biotechnology, Vol lo,1994
463
U.C. Banerjee Table 4. Effect of inorganic production by C. funata.’ inorganic nltrogen source (0.0496, expressed as nitrogen)
nitrogen
source
Maximum enzyme activity (NJ/ml)
NaNO, KNO,
on rifamycin
oxldase
Maximum enzyme productivity (IU/l.h)
17.9 12.0
124 125
WNO,),
12.0
125
(NH,&=, NH,NO,
25.0 16.3
260 113
(NH,),HPO,
14.0
146
W&PO, NH,CI
16.5 17.5 19.6
193 121 206
6.7 6.0
91 63
Urea Ammonium Ammonium Control
acetate oxalate
(no inorganic
nitrogen)
15
in promoting rifamycin oxidase synthesis (Table 3). Soybean and peanut meal, which are cheaper nitrogen sources than peptone, were both satisfactory, but peptone gave more consistent results. Peptone was therefore used in subsequent experiments. Ammonium sulphate was the most effective nitrogen source for promoting rifamycin oxidase synthesis (Table 4). Ammonium acetate and ammonium oxalate were inhibitory, presumably because of the oxalate and acetate moieties. Of other growth nutrients tested for promoting rifamycin oxidase production, yeast extract proved to be best (Figure 2).
Acknowledgements
102
The organism was grown in medium containing 10 g each of yeast extract, peptone and carboxymethylcellulose/l, at pH 6.5 and 28°C and for up to 6 days. l
Financial support from the Council of Scientific and Industrial Research and the Department of Biotechnology is acknowledged. The technical assistance of J.P. Srivastava and B. Saxena is greatly appreciated.
References U.C. 1993 Effect of glucose and carboxymethylcellulose on growth and rifamycin oxidase production by Curvularia lunata. Current Microbiology 26, 261-265. Banerjee, UC. & Srivastava, J.P. 1993 Effects of pH and glucose concentration on the production of rifamycin oxidase by Curvularia lunata in a batch reactor. Journal of Biotechnology
Banejee,
28,229-236.
10
80
120
Fermentation
Figure
2. Effect
by C. lunate,
of growth grown
factor, carboxymethylcellulose m-Yeast extract; A-beef infusion;
464
O-meat
factors
in medium
extract;
160
time (h)
on rifamycin containing
oxidase 10 g each
production
Han, M.H., Seong, B.L., Son, H.J. & Mheen, T.I. 1983 Rifamycin B oxidase from MonocilZium spp, a new type of diphenol oxidase. FEBS Letters 151,364O. Seong, B.L. & Han, M.H. 1982 A facile preparation of rifamycin derivatives by use of manganese dioxide. Chemistry Letters 5, 627-628. Seong, B.L., Son, H.J., Mheen, T.I. &Han, M.H. 1983 Microbial transformation of rifamycin B: a new synthetic approach to rifamycin derivatives. Journal of Antibiotics 36,1402-1404. Sensi, P. & Thiemann, J.E. 1967 Production of rifamycins. Progress in Industrial Microbiology 6,21-60. Vohra, R.M., Banerjee, U.C., Das, S. & Dube, S. 1989 Microbial transformation of rifamycin B: a new extracellular oxidase from Curvularia lunata. Biotechnology Letters, 11,851-854.
of growth
and
peptone/l, at pH 6.5 and 26%. extract; O-malt extract; O-liver A-control.
World Journal of Microbiology b Biotechnology, Vol lo,1994
(Received in revised form 2 March 1994; accepted 8 March 2994)